7[-2-Alkoxyamino(acetamido)]cephalosporin derivatives

ABSTRACT

Cephalosporins in a series having the formula ##STR1## wherein R 1  is alkyl containing 1-4 carbon atoms or a nontoxic pharmaceutically acceptable salt thereof, were synthesized and found to be potent antibacterial agents especially when in the form of the syn isomers essentially free of the corresponding anti isomer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The cephalosporins of the present invention in general possess the usualattributes of such compounds and are particularly useful in thetreatment of bacterial infections.

2. Description of the Prior Art

U.K. No. 1,399,086 disclosed antibiotic compounds of the general formula##STR2## (wherein R^(U) is phenyl; naphthyl; thienyl; furyl,benzothienyl; benzofuryl; pyridyl or any of these groups substituted byhalo (chloro, bromo, iodo or fluoro), hydroxy, lower alkyl, nitro,amino, loweralkylamino, diloweralkylamino, lower alkanoyl, loweralkanoylamino, lower alkoxy, lower alkylthio or carbamoyl; R^(b) islower alkyl; cycloalkyl containing 3-7 carbon atoms; carbocyclic orheterocyclic aryl lower alkyl or any of these groups substituted byhydroxy, carboxy, exterified carboxy, amido, cyano, alkanoyl, amino,substituted amino, halogen or lower alkoxy; and Y is selected fromacetoxy; a group of formula ##STR3## where R^(d) and n are as defined inclaim 19; a group of formula --SW where W is thiadiazolyl, diazolyl,triazolyl, tetrazolyl, thiazolyl, thiatriazolyl, oxazolyl, oxadiazolyl,benzimidazolyl, benzoxazolyl, triazolopyridyl, purinyl, pyridyl orpyrimidyl; an alkylthio group containing 1-4 carbon atoms; a group offormula --O.CO.R⁹ where R⁹ is an alkyl or alkenyl group containing 2-4carbon atoms; the group --O.CO.NH.(CH₂)_(m) D wherein m is an integer offrom 1-4 and D is chlorine, bromine, iodine or fluorine; and azido) andnon-toxic salts and esters thereof. Methods for the preparation of thestarting acids used to form the 7-substituent, including theirseparation into syn and anti isomers, are also described therein and inU.K. No. 1,404,221.

Presently issued U.S. Pat. Nos. 3,966,717 and 3,971,778 (with manynucleophilic substituents) contain at least part of the disclosure ofU.K. No. 1,399,086 as does U.S. Pat. No. 3,974,153. See also Farmdocabstracts 17270X, 19177X and 63415X and U.S. Pat. Nos. 4,024,134,4,007,174 and 4,066,762.

U.S. Pat. No. 3,974,153 claims compounds of the formula ##STR4## whereinR¹ is furyl, thienyl or phenyl; and R^(a) is C₁₋₄ alkyl, C₃₋₇ cycloalkylor phenyl; and a physiologically acceptable salt thereof.

With reference to cephalosporins in which the acetoxy group in the3-methyl substituent has been displaced by a thiol see U.S. Pat. No.3,741,965 for a review of the older art.

For examples of publications in the scientific literature see Ryan etal., Antimicrobial Agents and Chemotherapy, 9, 520-525 (1976) andO'Callaghan et al., ibid., 9, 511-519 (1976) and Norby et al., ibid., 9,506-510 (1976) regarding cefuroxime.

West Germany No. 2,451,480 (Farmdoc 32820W) apparently teaches a methodfor the preparation of thiols of the formula ##STR5## where R includesaminoethyl and there is further disclosure of that compound, methods forits preparation and its possible use to make a cephalosporin in U.K.Nos. 1,489,609, 1,489,610 and 1,461,948. See also Farmdoc 02692X,68777X, 23652Y, 27138Y, 30744Y, 69415Y, 72915Y and U.S. Pat. No.4,039,536.

SUMMARY OF THE INVENTION

One of the problems presently facing the medical profession at this timewas described by Arnold L. Smith, M.D. in an article titled Antibioticsand Invasive Haemophilus influenzae, N. Engl. J. Med., 294(24),1329-1331 (June 10, 1976) in which the opening sentence reads asfollows: "Recently the information service of the Center for DiseaseControl, the Medical Letter and the American Academy of Pediatrics havesounded the alert that invasive strains of Haemophilus influenzaeisolated throughout the United States have been found to be resistant toampicillin, many of the isolates being associated with treatmentfailure." His concluding paragraph reads: "The current situationportends a dismal future for the antibiotic treatment of invasive H.influenzae disease. An H. influenzae resistant to the second-line drug,chloramphenicol, has been described, and, more recently, an untypable H.influenzae resistant to chloramphenicol and tetracycline was isolatedfrom the throat of a four-year-old girl. Thus, both these currentlyefficacious agents may not be useful in the future."

A solution to this problem is provided by the present invention.

The present invention thus provides compounds having the formula##STR6## often written herein as ##STR7## wherein R¹ is alkyl containing1-4 carbon atoms and R² is hydrogen or a conventional, pharmaceuticallyacceptable, easily hydrolyzed ester forming group such as those setforth below and including the group having the formula ##STR8## whereinwhen W represents hydrogen, Z represents (lower)-alkanoyl, benzoyl,naphthoyl, furoyl, thenoyl, nitrobenzoyl, methylbenzoyl, halobenzoyl,phenylbenzoyl, N-phthalimido, N-succinimido, N-saccharino,N-(lower)alkylcarbamoyl, (lower)alkoxy, (lower)-alkylthio, phenoxy,carbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy,carbophenoxy, carbo-tert.-butoxy or (lower)alkylsulfonyl, and when Wrepresents carbalkoxy, Z represents carbalkoxy and, when W representsphenyl, Z represents benzoyl or cyano or wherein W and Z taken togetherrepresent 2-oxocycloalkyl containing 4 to 8 carbon atoms inclusive.

As set forth below in more detail the present invention also providesmetallic salts of these acids and acid addition salts of these esters inaddition to the "free acid" form, i.e. R is hydrogen, which existsprimarily as a zwitterion. The "free acid" or zwitterion form may alsobe converted by treatment with a nontoxic, pharmaceutically acceptableacid to an acid addition salt of the amino group, e.g. formate orhydrochloride. The stereochemistry of the bicyclic nucleus is that foundin cephalosporin C.

The compounds of the present invention are syn isomers or else aremixtures of syn and anti isomers containing at least 75% of the synisomer. Preferably such mixtures of isomers contain at least 90% of thesyn isomer and not more than 10% of the anti isomer. Most preferably thecompounds are syn isomers essentially free of the corresponding antiisomer.

The preferred embodiments of the present invention are the syn isomersof the compounds of Formula I wherein R¹ is methyl or ethyl and R² ishydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or5-indanyl.

Reference to the syn (cis) isomeric form refers to the configuration ofthe group OR¹ with respect to the carboxamido group.

The present invention also provides the process for the production ofthe antibacterial agents having the formula ##STR9## wherein R¹ is alkylcontaining 1-4 carbon atoms which comprises reacting a compound of theformula ##STR10## or a salt or easily hydrolyzed ester or Schiff base aswith benzaldehyde or salicylaldehyde thereof (including, but not limitedto, those of U.S. Pat. No. 3,284,451 and U.K. No. 1,229,453 and any ofthe silyl esters described in U.S. Pat. No. 3,249,622 for use with7-aminopenicillanic acid and used in Great Britain No. 1,073,530 andparticularly the pivaloyloxymethyl, acetoxymethyl, methoxymethyl,acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyland 5-indanyl esters) thereof with an organic monocarboxylic acidchloride or a functional equivalent thereof as an acylating agent.

Such functional equivalents include the corresponding acid anhydrides,including mixed anhydrides and particularly the mixed anhydridesprepared from stronger acids such as the lower aliphatic monoesters ofcarbonic acid, or alkyl and aryl sulfonic acids and of more hinderedacids such as diphenylacetic acid. In addition, an acid azide or anactive ester or thioester (e.g. with p-nitrophenyl, 2,4-dinitrophenol,thiophenol, thioacetic acid) may be used or the free acid itself may becoupled with compound II after first reacting said free acid withN,N'-dimethylchloroformiminium chloride [cf. Great Britain No. 1,008,170and Novak and Weichet, Experientia XXI, 6, 360 (1965)] or by the use ofenzymes or of an N,N'-carbonyldiimidazole or an N,N'-carbonylditriazole[cf. South African patent specification No. 63/2684] or a carbodiimidereagent [especially N,N'-dicyclohexylcarbodiimide.N,N'-diisopropylcarbodiimide orN-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; cf. Sheehan and Hess.J. Amer. Chem. Soc., 77, 1967 (1955)], or of alkylylamine reagent [cf.R. Buijle and H. G. Veihe, Angew, Chem. International Edition 3, 582,(1964)] or of an isoxazolium salt reagent [cf. R. B. Woodward, R. A.Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)], or of aketenimine reagent [cf. C. L. Stevens and M. E. Munk, J. Amer. Chem.Soc., 80, 4065 (1958)] or of hexachlorocyclotriphosphatriazine orhexabromocyclotriphosphatriazine (U.S. Pat. No. 3,651,050) or ofdiphenylphosphoryl azide [DPPA; J. Amer. Chem. Soc., 94, 6203-6205(1972)] or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No.18, pp. 1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron LettersNo. 49, pp. 5047-5050 (1972)]. Another equivalent of the acid chlorideis a corresponding azolide, i.e., an amide of the corresponding acidwhose amide nitrogen is a member of a quasiaromatic five membered ringcontaining at least two nitrogen atoms, i.e., imidazole, pyrazole, thetriazoles, benzimidazole, benzotriazole and their substitutedderivatives. As an example of the general method for the preparation ofan azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acidin equimolar proportions at room temperature in tetrahydrofuran,chloroform, dimethylformamide or a similar inert solvent to form thecarboxylic acid imidazolide in practically quantitative yield withliberation of carbon dioxide and one mole of imidazole. Dicarboxylicacids yield diimidazolide. The by-product, imidazole, precipitates andmay be separated and the imidazolide isolated, but this is notessential. The methods for carrying out these reactions to produce acephalosporin and the methods used to isolate the cephalosporin soproduced are well known in the art.

Mention was made above of the use of enzymes to couple the free acidwith compound II. Included in the scope of such processes are the use ofan ester, e.g. the methyl ester, of that free acid with enzymes providedby various microorganisms, e.g. those described by T. Takahashi et al.,J. Amer. Chem. Soc., 94(11), 4035-4037 (1972) and by T. Nara et al., J.Antibiotics (Japan) 24(5), 321-323 (1971) and in U.S. Pat. No.3,682,777.

For the coupling of the organic carboxylic acid as described above withcompound II (or a salt or preferably an easily hydrolyzed ester ofSchiff base, as with benzaldehyde, thereof) it is also convenient andefficient to utilize as the coupling agent phosphonitrilic chloridetrimer (J. Org. Chem., 33(7), 2979-81, 1968) orN-ethoxy-1,2-dihydroquinoline (EEDQ) as described in J. Amer. Chem.Soc., 90, 823-824 and 1652-1653 (1968) and U.S. Pat. No. 3,455,929. Thereaction is preferably carried out at 30°-35° C. in benzene, ethanol ortetrahydrofuran using about equimolar quantities of all three reagentsfollowed by conventional isolation and removal by conventional methodsof any blocking groups present.

An additional process of the present invention comprises the preparationof the compounds of the present invention by the displacement of the3-acetoxy group of a 7-acylaminocephalosporanic acid (prepared bysubstituting 7-aminocephalosporanic acid for the3-thiolated-7-aminocephalosporanic acids in the acylation proceduresdescribed herein and elsewhere reported) with a thiol HSR³ having theformula ##STR11## wherein B is hydrogen or a protecting group for aprimary amine and then removing the protecting group if any is present,as on the aminoethyl group and/or the carboxyl group.

The displacement of such a 3-acetoxy group with such a thiol may beaccomplished in solution as in water or aqueous acetone at a temperatureof at least room temperature and preferably within the range of about50° to 100° C. in the presence of a mild base such as sodiumbicarbonate, e.g. preferably near neutrality such as at about pH 6. Anexcess of the thiol is preferably employed. The reaction product isisolated by careful acidification of the reaction mixture followed byextraction with a water-immiscible organic solvent. As noted above, thepreparation of many other 7-acylamidocephalosporanic acids is describedin the patent and scientific literature, e.g. in U.S. Class 260-243 C.

The salts of the compounds of this invention include the nontoxiccarboxylic acid salts thereof, including nontoxic metallic salts such assodium, potassium, calcium and aluminum, the ammonium salt andsubstituted ammonium salts, e.g. salts of such nontoxic amines asL-lysine, arginine, histidine, trialkylamines including triethylamine,procaine, dibenzylamine, N-benzylbeta-phenethylamine, 1-ephenamine,N,N'-dibenzylethylenediamine, dehydroabietylamine,N,N'-bis-dehydroabietylethylenediamine, and other amines which have beenused to form salts with benzylpenicillin and also nontoxic acid additionsalts (of the primary amino group) thereof including the mineral acidaddition salts such as the hydrochloride, hydrobromide, hydroiodide,sulfate, sulfamate and phosphate and the organic acid addition saltssuch as the maleate, acetate, citrate, oxalate, succinate, benzoate,tartrate, fumarate, malate, mandelate, ascorbate, formate and the like.

The preferred esters of the cephalosporins of the present invention arethe pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl andphenacyl esters. All are useful intermediates in the production of thecephalosporin having a free carboxyl group.

As indicated above, these five esters of 7-aminocephalosporanic acid areeach prepared by known methods. One excellent procedure is that of U.S.Pat. No. 3,284,451 in which sodium cephalothin is esterified by reactionwith the corresponding active chloro or bromo compound (e.g. phenacylbromide, chloroacetone, chloromethyl ether, pivaloyloxymethyl chloride[also called chloromethyl pivalate], acetoxymethyl chloride) and thenthe thienylacetic acid sidechain is removed enzymatically as in the samepatent or chemically as in U.S. Pat. No. 3,575,970 and in Journal ofAntibiotics, XXIV (11), 767-773 (1971). In another good method thetriethylamine salt of 7-aminocephalosporanic acid is reacted directlywith the active halogen compound, as in United Kingdom No. 1,229,453.

These esters of 7-aminocephalosporanic acid are then reacted with thenucleophile HSR³ in the same manner as is illustrated herein for7-aminocephalosporanic acid itself. The 3-thiolated ester of7-aminocephalosporanic acid is then coupled with the organic carboxylicacid as before.

The ester of the cephalosporin so obtained is, if not used per se,converted to its free acid and, if desired, any salt by removal of theesterifying group, as by aqueous or enzymatic hydrolysis (as with humanor animal serum) or acidic or alkaline hydrolysis or by treatment withsodium thiophenoxide as taught in U.S. Pat. No. 3,284,451 and, in thepenicillin series, by Sheehan et al., J. Org. Chem. 29(7), 2006-2008(1964).

In another alternative synthesis, the 3-thiolated 7-aminocephalosporanicacid is prepared as described herein and then acylated at the 7-aminogroup and finally esterified, as by reaction of the appropriate alcoholwith the acid chloride prepared, for example, by reaction of the finalcephalosporin with thionyl chloride or by other essentially acidicesterification procedures.

In the treatment of bacterial infections in man, the compounds of thisinvention are administered parenterally in an amount of from about 10 to90 mg./kg./day and preferably about 14 to 50 mg./kg./day in divideddosage, e.g. two to four times a day. They are administered in dosageunits containing, for example, 125, 250 or 500 mg. of active ingredientwith suitable physiologically acceptable carriers or excepients. Thedosage units are in the form of liquid preparations such as solutions orsuspensions and preferably are aqueous solutions of a sodium, potassium,hydrochloride or formate salt which are injected intravenously orintramuscularly or by continuous or intermittent infusion inconcentrations of about 125-500 mgm./ml., and preferably, 250 mgm./ml.as is customary in therapy with cephalosporin antibiotics.

STARTING MATERIALS 2-Furoylcyanide

To a suspension of 26.1 g. (0.4 mole) of ground potassium cyanide in 300ml. of acetonitrile at 5° C. was added 26.1 g. (0.2 mole) of α-furoylchloride while keeping the temperature below 8° C. The mixture wasstirred in the cold for 15 minutes then heated at reflux for 30 minutes.The reaction was cooled, filtered and the acetonitrile was removed at 15mm. (steam-bath) leaving 24.5 g. of a dark oil which was used withoutfurther purification. An infrared spectrum showed a nitrile band at 2265cm⁻¹.

2-Furaneglyoxylic Acid

The 24.5 g. of crude 2-furoylcyanide was mixed with 160 ml. concentratedhydrochloric acid at 25° C. with intermittent stirring. The reaction wasstored for 24 hours at 25° C. and diluted with 80 ml. of water. Thereaction was stirred for 5 minutes and filtered. The filtrate wassaturated with sodium chloride and extracted with 5×120 ml. of 1:1ether-ethyl acetate solution. The extracts were combined, dried overanhydrous magnesium sulfate and evaporated at 30° C. (15 mm.) to give abrownish-orange solid. The solid was dissolved in methanol, treated withcharcoal and evaporated under reduced pressure (15 mm.) to dryness toyield 17 g. of the acid.

The product was recrystallized from toluene to give 11.5 g. (m.p. 76°C.). The IR and NMR spectra were consistent for the structure.

2-Methoxyimino-2-furylacetic Acid

To a solution of 4.5 g. (0.032 mole) of 2-furaneglyoxylic acid in 40 ml.of 50% alcohol and 3.1 g. (0.037 mole) of methoxyamine hydrochloride in6 ml. water at 20° C. was added dilute sodium hydroxide solution to pH4-5. The solution was stirred at pH 4-5 at 25° C. for 24 hours. Thealcohol was removed under reduced pressure (15 mm.) and the solution wasadjusted to pH 7-8 with 50% sodium hydroxide solution. The reaction waswashed with 3×50 ml. of ether and the aqueous layer was adjusted to pH1.9 using concentrated hydrochloric acid. The mixture was extracted with5×50 ml. of ethyl acetate. The ethyl acetate extracts were combined,washed with brine, dried over anhydrous magnesium sulfate and evaporatedunder reduced pressure (15 mm.) to an oil which was cooled for one hourin an ice bath. The product was slurried with Skellysolve-B andcollected to yield 3.1 g. of yellow crystals, m.p. 78° C. An analyticalsample was recrystallized from toluene, dried for 16 hours in vacuo overP₂ O₅ at 25° C. The IR and NMR spectra were consistent for thestructure.

Anal. Calc'd for C₇ H₇ NO₄ : C, 49.65; H, 4.17; N, 8.28. Found: C,49.30; H, 4.21; N, 8.37.

"Skellysolve-B" is a petroleum ether fraction of b.p. 60°-68° C.consisting essentially of n-hexane.

2-Ethoxyiminofurylacetic Acid

7.85 g. (0.056 mole) of furyl-2-glyoxylic acid (2-furaneglyoxylic acid)was dissolved in 100 ml. of water and adjusted to pH 7 with 50% sodiumhydroxide. 6.83 g. of ethoxyamine hydrochloride in 10 ml. of water wasadded, while keeping the pH at 4-5. The reaction was diluted with 25 ml.of alcohol, stirred 3 hours at room temperature and then filtered. Thealcohol was removed at 35° C. (15 mm.) and the aqueous portion wasadjusted with dilute sodium hydroxide solution to pH 7-8 and then waswashed with ether and the washes were discarded. The aqueous fractionwas adjusted with 6 N hydrochloric acid to pH 1.5 and extracted into3×80 ml. of ethyl acetate. The acetate fractions were combined, washedwith brine and reduced in volume at 35° C. (15 mm.) to an oil. The oilwas cooled in an ice bath, triturated with "Skellysolve-B", collectedand dried over P₂ O₅ in vacuo at 25° C. Yield: 4.8 g., m.p. 83°-85° C.The IR and NMR were consistent for the structure.

Anal. Calc'd for C₈ H₉ NO₄ : C, 52.46; H, 4.95; N, 7.65. Found: C,52.22; H, 4.94; N, 7.60.

Sodium α-Ethoxyimino-α-(2-furyl)acetate

To 50 ml. of methanol was added 250 mg. (0.0109 mole) of metallic sodiumand stirred until all the sodium had dissolved. This sodium methoxidesolution was treated with 2.0 g. (0.0109 mole) ofα-ethoxyimino-α-(2-furyl)acetic acid dissolved in 10 ml. of methanol andstirred at room temperature for one hour. The methanol was removed at40° C. (15 mm.) and the product was dried in vacuo over P₂ O₅ at 25° C.to yield 2.22 g. white solid, m.p. decomp. >240° C. The IR and NMR wereconsistent for the structure. ##STR12##

2-Furoylcyanide 1

To a suspension of 78.3 g. of powdered potassium cyanide in 900 ml.acetonitrile at 5° C. was added 59.25 ml. (68.5 g.) of α-furoyl chloridewith vigorous stirring while keeping the temperature at 4°-8° C. Themixture was stirred at 4°-8° C. for 15 minutes and then heated at refluxfor 30 minutes. The mixture was cooled to 23°-25° C., filtered andwashed with 50 ml. of acetonitrile which was added to the filtrate. Theacetonitrile was removed at 60° C. (15 mm.) leaving 51 g. of 1 as a darkoil. An IR spectrum showed a nitrile band at 2265 cm⁻¹ and an NMRspectrum showed a ratio of approximately 70/30 of product 1 furoic acid.The crude product 1 was used without further purification (49% yield ofproduct).

Furyl-2-glyoxylic Acid 2

The 51 g. of crude 2-furoyl cyanide 1 was mixed with 500 ml.concentrated hydrochloric acid at 25° C. The reaction was stirred for 24hours at 25° C. and then diluted with 240 ml. of water. The mixture wasstirred for 5 minutes and filtered. The black filtrate was saturatedwith sodium chloride and extracted with 6×500 ml. of 1:1 ether-ethylacetate solution. (Note: Initially the extractions were difficult due tothe inability to see the separation of two black phases. As additionalether-ethyl acetate extractions were run the task was simplified.) Thesolvent extracts were combined and evaporated to dryness at 60° C. (15mm.). The resultant solid was dissolved in 600 ml. ether, (Note: Use ofalcohols should be avoided at this point as esters may form), treatedwith 10 g. of charcoal ("Darko-KB"), filtered after stirring for 0.5hour and evaporated to dryness at 50° C. (15 mm.) to yield 46.6 g. of 2as a light, tan-colored acid. This product 2 was found to contain aratio of approximately 56/44 of product 2/furoic acid. This representeda 63% yield of product 2.

Purification was accomplished by dissolving the above crude product 2 inH₂ O (50 mg./ml.), titrating to pH 2.8 with HCl and extracting with2×200 ml. of ethyl acetate. Evaporation of the ethyl acetate extractsgave 85% furoic acid and 15% product 2. The pH 2.8 aqueous phase wasadjusted to pH 0.8 (HCl) and extracted with 2×200 ml. ethyl acetate. Theorganic extracts were combined and washed with 50 ml. H₂ O. The organicphase was evaporated at 50° C. (15 mm.) yielding a solid with a ratio ofapproximately 86/14 of product 2/furoic acid. This solid was thenrecrystallized by dissolving the product 2 in toluene at 50 mg./ml. at80° C., decanting, and leaving to crystallize at room temperature for 18hours, yielding 13.3 g. of pure acid 2 by NMR. This represented a 51%yield in the purification and recrystallization step and an overallyield from the 2-furoyl chloride to the pure furyl-2-glyoxylic acid 2 of16%.

Syn-α-methoxyiminofurylacetic Acid 3

A solution of 4.5 g. of furyl-2-glyoxylic acid 2 in 40 ml. of 50%ethanol was titrated to pH 6 with 1 N sodium hydroxide and then 3.1 g.of methoxyamine.HCl in 6 ml. of H₂ O at 20° C. was added. The solutionwas titrated to a constant pH 4.9 and stirred at pH 4.9 for 24 hours at20°-23° C. The ethanol was then removed at 50° C. (15 mm.) and theresidual aqueous solution was titrated to pH 8 with 50% sodium hydroxideand washed with 3×50 ml. ether (pH adjusted to 8 after each wash). Theaqueous layer was titrated to pH 1.9 with concentrated HCl and extractedwith 5×50 ml. ethyl acetate with the pH readjusted to 1.9 after eachextraction. The ethyl acetate extracts were combined and evaporated to asolid 3 at 50° C. (15 mm.). This solid was then slurried with 75 ml. of"Skellysolve B". The suspension was filtered and the solids wereredissolved in 16 ml. of toluene at 80° C. The hot solution was decantedand left to crystallize at 20°-23° C. for 18 hours to yield 1.17 g. 3(22% yield of product). The NMR was clean and consistent for thestructure 3 with a trace of anti isomer present.

Sodium Syn-α-methoxyiminofurylacetate 4

To 40 ml. of methanol was added 0.16 g. of sodium. The mixture wasstirred until all of the sodium dissolved and then decanted. Theresulting sodium methoxide solution was cooled to 3° C. and 1.12 g. ofsyn-α-methoxyiminofurylacetic acid 3 in 7.8 ml. of methanol was added.The solution was stirred for 10 minutes at room temperature. The solventwas evaporated at 40° C. (15 mm.). The residue 4 was dried by azeotropicdistillation with 3×20 ml. of benzene at 40° C. (15 mm.). The product 4was dried for 18 hours at 23° C. under high vacuum (0.7 mm.) over P₂ O₅yielding 1.25 g. (99% yield of product). The NMR showed this product 4to be clean and consistent for the structure with 0.15 mole methanol anda trace of anti isomer.

To 0.63 g. of sodium syn-α-methoxyiminofurylacetate 4 suspended in 25ml. of benzene was added four drops of dry dimethylformamide and 0.31ml. (1.1 eq.) of oxalyl chloride. This mixture was stirred for 40minutes at 20°-23° C. The benzene was removed at 35° C. (15 mm.) toprovide the acid chloride 5 as a gummy residue.

A. 2-Aminoethylacetamide ##STR13##

To 1915 ml. (1721.6 g.; 28.64 M.) ethylene diamine was added 933 ml.(841 g.; 9.5 M.) ethyl acetate. The reaction mixture was allowed tostand at room temperature for 5 days. The unreacted ethylene diamine wasremoved at 70° C. (˜15 mm). The main fraction was distilled at 110°-119°and 0.8 mm to yield 642 g. (66%) (m.p.≅43° C.) of 2-aminoethylacetamide.

B. 2-Isothiocyanoethylacetamide ##STR14##

To a stirred three-phase system of 84 g. NaHCO₃ in 225 ml. water and15.32 g. (0.15 M.) 2-aminoethylacetamide in 247 ml. CHCl₃ was addeddropwise 14.3 ml. (21.56 g.; 0.187 M.) thiophosgene in 160 ml. CHCl₃ atroom temperature. The orange mixture was stirred for 18 hours at roomtemperature (19°-20° C.).

The resulting yellow mixture was filtered to remove the excess NaHCO₃and the two layers were separated. The water phase was washed twice with50 ml. CHCl₃. The combined organic phases were washed with saturatedsodium chloride solution, dried over anhydrous MgSO₄ and evaporated at38°-45° C. (15 mm) to give 2-isothiocyanoethylacetamide as an oil; 27.5g. (contaminated with CHCl₃). Weight corrected for 0.6 M CHCl₃ from NMRwas 18 g. (83% yield CHCl₃ free).

C. 1-Acetamidoethyl-5-mercaptotetrazole ##STR15##

To a stirred solution of 11.3 g. (0.1738 M.) NaN₃ (CAUTION!) in 200 ml.water at 65° C. under N₂ was added dropwise 17 g. (about 0.1248 M.)2-isothiocyanoethylacetamide in 20 ml. CHCl₃. The resulting mixture wasstirred for 21/2 hours at 75° C. and stored for 20 hours at roomtemperature.

The filtered solution was cooled and acidified to pH 1.5 with 6 N HCl.The sample was extracted three times with 100 ml. ethyl acetate. TheEtOAc was washed with 50 ml. of saturated sodium chloride solution,dried over anhydrous MgSO₄ and evaporated at 40° C. (15 mm.) to a yellowsolid. The solid 1-acetamidoethyl-5-mercaptotetrazole was trituratedwith ether, filtered, dried in vacuo and found to weigh 4.78 g. (21%yield).

D. 2-Isothiocyanoethylacetamide ##STR16## NOTE: The procedure below wasrun simultaneously in three separate 2-liter flasks.

To a stirred three-phase system of 168 g. NaHCO₃ in 450 ml. water and30.64 g. (0.3 M.) 2-aminoethylacetamide in 494 ml. CHCl₃, was addeddropwise 28.6 ml. (43.1 g.; 0.373 M.) thiophosgene in 320 ml. CHCl₃ atroom temperature. The orange mixture was stirred for 18 hours at roomtemperature.

The resulting yellow mixture was filtered to remove the excess NaHCO₃and the two layers were separated. The water phase was washed twice with100 ml. CHCl₃. The combined organic phases were washed with 75 ml.saturated sodium chloride solution, dried over anhydrous MgSO₄ andevaporated at 40° C. (15 mm.) to oily crystals of2-isothiocyanoethylacetamide.

    ______________________________________                                        Yield:        I - 59.7 g. (26% CHCl.sub.3 →44.2 g.)                                  II - 49.3 g. (15% CHCl.sub.3 →41.7 g.)                                III - 51.6 g. (15% CHCl.sub.3 →43.9 g.)                                TOTAL = 129.8 g. (100%)                                          ______________________________________                                    

E. 1-Acetamidoethyl-5-mercaptotetrazole ##STR17##

To a stirred solution of 87.6 g. (1.3477 M.) NaN₃ (CAUTION!) in oneliter water at 65° C. under N₂ was added dropwise 126.7 g. (0.8786 M.)2-isothiocyanoethylacetamide in 150 ml. CHCl₃. The resulting mixture wasstirred for three hours 10 minutes at 70°-72° C. and then cooled to 15°C., filtered and acidified to pH 1.5 with concentrated HCl (CAUTION!hydrozoic acid is liberated). The sample was extracted four times with500 ml. ethyl acetate. The EtOAc was washed with 300 ml. saturatedsodium chloride solution, dried over anhydrous MgSO₄ and evaporated at40° C. (15 mm.) to a yellow solid.

The solid was triturated with a minimum amount of ether, filtered anddried in vacuo over P₂ O₅ for 5 days to give 61.3 g. of1-acetamidoethyl-5-mercaptotetrazole (37% yield).

F. 1-Aminoethyl-5-mercaptotetrazole Hydrochloride ##STR18##

A mixture of 61.3 g. (0.3274 M.) 1-acetamidoethyl-5-mercaptotetrazoleand 313 ml. 6 N HCl (196 mg./ml.; 1.888 M.) was heated at reflux for 2hours. The resulting solution was treated with 10 g. carbon andevaporated at 45° C. (15 mm.) to a solid which was triturated with 90ml. glacial acetic acid, washed with 250 ml. ether and dried in vacuoover P₂ O₅ to give 45.6 g. (77% yield) of1-aminoethyl-5-mercaptotetrazole hydrochloride.

G. 2-Carboethoxyethyl Isothiocyanate ##STR19##

β-Alanine (178.18 g., 2 M.) was slurried in absolute ethanol (500 ml.)then gassed vigorously with HCl for 41/2 hours keeping the temperaturebetween 20°-30° C. with an ice bath. The resulting solution was thenevaporated to an oil on a rotovac (50° C., 5 mm.) and azeotroped to adry oil with 100 ml. ethyl acetate (50° C., 5 mm.). The dry oil was thencrystallized by slurrying in 200 ml. ether. The crystals were collectedby filtration, washed with 30 ml. of ether and dried in a desiccatorover P₂ O₅ using house vacuum for 18 hours to yield 280.4 g. (91%)β-alanine ethyl ester hydrochloride; m.p. <50° C. The NMR was consistentfor the structure with about 10% unreacted β-alanine.

β-Alanine ethyl ester hydrochloride (171.3 g.; 1.11 M.) andtriethylamine (TEA) (312 ml.) and 700 ml. methylene chloride were mixedtogether then cooled to -10° C. in an ice-acetone-salt bath. The CS₂ (67ml.; 1.11 M.) in 250 ml. CHCl₃ was added over a 11/2 hour period at atemperature of -12° to -5° C. The temperature was then allowed to warmto 10° C. and stirred at this temperature for 10 minutes. The mixturewas then cooled to 0° to 5° C. and 105.7 ml. (1.11 M) ethylchloroformate in 100 ml. CHCl₃ added dropwise during a 40-minute period.The mixture was stirred 30 minutes at room temperature then cooled to 0°C. TEA (156 ml.; 1.11 M) was added dropwise at 0° C. over 1/2 hour thenstirred 3 hours at room temperature.

The mixture was stirred with 450 ml. water, separated and the organicphase was washed twice with 450 ml. 2 N HCl, twice with 450 ml. 5%NaHCO₃, twice with 360 ml. water, dried over Na₂ SO₄ and then evaporatedto give 148.5 g. (84% yield) of 2-carboethoxyethyl isothiocyanate as anoil.

H. 1-Carboxyethyl-5-mercaptotetrazole ##STR20## NOTE: Caution is to betaken with NaN₃ ; dust may be toxic (fatal)

110.5 g. (1.7 M.) NaN₃ was weighed out cautiously in the hood anddissolved in one liter water in a 2-liter R.B. 3-necked flask under N₂and heated to 60° C. Using a dropping funnel, 175.0 g. (1.1 M.)2-carboethoxyethyl isothiocyanate in 180 ml. "Skellysolve B" (mixedlower alkanes) was added and the mixture was heated to 70° C. andstirred at 70°-72° C. for 21/2 hours.

The mixture was cooled to 30° C., titrated with concentrated NaOH to pH11.5, heated to 70° C. for 40 minutes (pH 10-11.5) then cooled to 15°C., acidified to pH 2 with concentrated hydrochloric acid and extractedfour times with 500 ml. ethyl acetate. The ethyl acetate extracts werewashed with 400 ml of water dried over Na₂ SO₄ and the resulting clear,yellow solution was evaporated to dryness, slurried with methylenechloride (MeCl₂), collected by filtration and washed with MeCl₂ to give82 g. (43% yield) of 1-carboxyethyl-5-mercaptotetrazole.

I. 1-Carboxyethyl-5-diphenylmethylthiotetrazole ##STR21##

To a solution of 82 g. (0.47 M.) 1-carboxyethyl-5-mercaptotetrazole in690 ml. dimethylformamide (DMF) was added 65 g. (0.47 M.) K₂ CO₃(anhydrous) and 116 g. (0.47 M.) benzhydryl bromide and the mixture wasstirred 19 hours at room temperature.

The mixture was then diluted with two liters water, acidified withconcentrated hydrochloric acid to pH 2 and extracted into ethyl acetate(3×1 L). The EtOAc was washed with 150 ml. water, dried with MgSO₄,filtered, evaporated to an oil, crystallized with one liter water,collected by filtration, washed with 500 ml. water and vacuum-dried at50° C. to give 144.5 g. (90% yield) of1-carboxyethyl-5-diphenylmethylthiotetrazole.

J. 1-Benzyloxycarbonylaminoethyl-5-diphenylmethylthiotetrazole ##STR22##

To a solution of 50.0 g. (0.1469 M.)1-carboxyethyl-5-diphenylmethylthiotetrazole in 500 ml. tetrahydrofuran(THF) at 0°-5° C. was added 20.5 ml. TEA and 15.4 ml. (1.1 eq.)ethylchloroformate. Stirred for 20 minutes and 9.6 g. (0.1469 M.) NaN₃(CAUTION-) in 250 ml. water was added slowly with stirring for 2 hoursat 0°-5° C. (two layers). The solution was diluted with 500 ml. waterand the THF evaporated at 30° C. (15 mm.). The mixture was thenextracted three times with 500 ml. ether. Benzene (500 ml.) was addedand the solution was dried over anhydrous MgSO₄. After filtering awaythe MgSO₄, 30.4 ml. (0.2938 M.) benzyl alcohol was added and the etherwas removed by fractional distillation. The residual benzene solutionwas then heated at reflux for two hours (75°-80° C.) and the benzene wasthen removed at 45° C. (15 mm.) to leave an oil which was dissolved in500 ml. CCl₄ and stored at 4° C. for three days to give 9.4 g. (15%yield) solid 1-benzyloxycarbonylaminoethyl-5-diphenylmethylthiotetrazolewhich was collected by filtration.

K. 1-Benzyloxycarbonylaminoethyl-5-mercaptotetrazole ##STR23##

TEA (63 ml.) was added to 9.4 g. (0.0211 M.) of1-benzyloxycarbonylaminoethyl-5-diphenylmethylthiotetrazole and 9.4 g.(0.1 M.) phenol and left standing at room temperature for 31/2 hours.The TFA was then evaporated off at 40° C. (15 mm.). The resulting oilyresidue was added to 63 ml. of a 5% NaHCO₃ solution and 30 ml. of etherand titrated with concentrated NaOH to pH 8.2. The phases were separatedand the aqueous water phase was titrated to pH 2 with concentratedhydrochloric acid, extracted twice with 60 ml. ether and the ether wasevaporated to give an oil. The oil was azeotroped with ethyl acetate andcrystallized by scratching with a glass rod to give1-benzyloxycarbonylaminoethyl-5-mercaptotetrazole which was washed with100 ml. "Skellysolve-B". 3.67 g. (62% yield) was collected byfiltration.

L. 1-Aminoethyl-5-mercaptotetrazole.HBr ##STR24## 18.25 ml. 30% HBr inglacial acetic acid solution was added to 3.65 g. (0.013 M.)1-benzyloxycarbonylaminoethyl-5-mercaptotetrazole. There was immediateevolution of CO₂ and a complete solution formed followed by aprecipitate upon stirring for one hour. The precipitate was collected byfiltration, washed with 30% HBr-HAc solution and then with ether (250ml.) to give a first crop of 1-aminoethyl-5-mercaptotetrazole.HBr (2.08g.; 72% yield) containing about 12% benzyl bromide by NMR.

The filtrate and washes from above were left standing at roomtemperature for 18 hours to give a second crop of1-aminoethyl-5-mercaptotetrazole.HBr which was collected by filtrationand washed with ether. Yield: 0.8 g. (27%) containing about 4% benzylbromide by NMR. Total yield of pure compound was 89%.

M. 1-(2'-t-Butoxycarbonylaminoethyl)-5-mercaptotetrazole ##STR25##

1-Aminoethyl-5-mercaptotetrazole.HCl, 45.6 g. (0.2510 M.) was dissolvedin 200 ml. dimethylsulfoxide (DMSO) 64 ml. (57.8 g.; 0.502 M.)1,1,3,3-tetramethylguanidine and 51.4 ml. (53.6 g.; 0.2761 M.)t-butylphenylcarbonate were added and the solution was stirred at roomtemperature for 42 hours.

After stirring the solution was diluted with 1500 ml. water and thenextracted with 900 ml. ethyl acetate and the ethyl acetate wasdiscarded. The aqueous layer was then stirred with 900 ml. fresh ethylacetate and acidified to pH 2 with concentrated hydrochloric acid. Thelayers were separated and the aqueous phase was extracted again with 900ml. ethyl acetate. The two ethyl acetate extracts were combined andevaporated at 60° C. (15 mm.) for 1 hour to give 47.9 g. of an oil.

The product was crystallized by slurrying with 200 ml. water collectedby filtration, washed with 400 ml. water and dried at 30° C. (15 mm.)for 18 hours to give 36.1 g. (59% yield) of1-(2'-t-butoxycarbonylaminoethyl)-5-mercaptotetrazole.

N.7-Amino-3-(1-[2'-t-butoxycarbonylaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicAcid ##STR26##

To 900 ml. pH 7 phosphate buffer heated to 45° C. with N₂ bubblingthrough, the following were added in three minutes at 45° C.: 39.1 g.(0.1438 M.) 7-aminocephalosporanic acid; 5.5 g. NaHSO₃ ; 38.8 g. (0.1582M.) 1-(2'-t-butoxycarbonylaminoethyl)-5-mercaptotetrazole. The nitrogenwas then shut off and the mixture was heated to 56° C., titrated to pH6.6 with NaHCO₃ (about 35 g.); and kept at 56° C. for 4 hours (pH6.3-6.6). After 4 hours at 56° C. the mixture was cooled to 5° C.,titrated to pH 5.0 and the resultant solid product was collected byfiltration and washed with 100 ml. water to give 35.8 g. (54% yield):[KF(H₂ O)--32.52% found; 53 g. discounted for 32.5% water] of 7-amino-3-(1-[2'-t-butoxycarbonylaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 A.7-(α-Methoxyiminofurylacetamido)-3-(1-[2'-t-butoxycarboxyaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicAcid ##STR27##

Sodium α-methoxyiminofuryl acetate (0.63 g.; 3.3 mM.) was suspended in25 ml. benzene and treated with 4 drops of dry dimethylformamide (DMF).A total of 0.31 ml. (0.46 g., 1.1 eq.) of oxalyl chloride was added andthe solution was stirred for 40 minutes at 20° C.

The benzene was removed at 35° C. (15 mm.) and the acid chloride (thegummy residue) was dissolved in 10 ml. acetone (NaCl insoluble). Asolution of 1.2 g. (2.6 mM.)7-amino-3-(1-[2'-t-butoxycarboxyaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid and 0.7 g. NaHCO₃ (pH 7.0) and 2.5 ml. acetone in 20 ml. water wascooled to 3° C. The acetone solution of the above acid chloride wasadded and the reaction was stirred for 40 minutes at 20° C. (pH 7.0).

The acetone was removed at 40° C. (15 mm.) and the aqueous solution wastitrated to pH 1.8 with hydrochloric acid (4 N). The product wasextracted three times with 60 ml. ethyl acetate, backwashed with 50 ml.water and azeotroped to dryness at 30° C. (15 mm.). The residue wastriturated with 50 ml. ether, collected by filtration and dried in vacuo18 hours to give 0.88 g. (55% yield) of7-(α-methoxyiminofurylacetamido)-3-(1-[2'-t-butoxycarboxyaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

Analysis Calculated for C₂₃ H₂₈ N₈ O₈ S₂.H₂ O (0.16 M. ether): KF(H₂ O),2.82; C, 44.46; H, 4.99; N, 17.55; S, 10.04. Found: KF(H₂ O), 2.41; C,45.11; H, 4.93; N, 17.00; S, 9.64.

B.7-(α-Methoxyiminofurylacetamido)-3-(1-[2'-aminoethyl]-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicAcid ##STR28##

A solution of 400 mg. (0.6571 mM.)7-(α-methoxyiminofurylacetamido)-3-(1-[2'-t-butoxycarboxyaminoethyl]-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid in 10 ml. 97% HCOOH (40 mg./ml.) was stirred for 2 hours at roomtemperature. The excess HCOOH was then evaporated at 30° C. (15 mm.).The residue was azeotroped to dryness three times with 4 ml. ethylacetate. The residue was triturated with ether, collected by filtrationand washed with ether to give 0.36 g. (contaminated with ether, ethylacetate) of7-(α-methoxyiminofurylacetamido)-3-(1-[2'-aminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

IR and NMR consistent for structure: about 0.4 mole formic acid, about0.5 mole ethyl acetate and about 0.3 mole ether per mole of product.

Analysis calculated for C₁₈ H₂₀ N₈ O₆ S₂.H₂ O [0.5 M HCOOH; 0.5 M ethylacetate; 0.3 M ether]: KF(H₂ O), 2.92; C, 42.32; H, 4.90; N, 18.19; S,10.41. Found: KF(H₂ O), 3.36; C, 42.47; H, 4.63; N, 18.39; S, 10.07.

EXAMPLE 2 A.7-Amino-3-(1-[2'-t-butoxycarbonylaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicAcid ##STR29##

To 258 ml. pH 7 phosphate buffer heated to 45° C. with N₂ bubblingthrough, the following were added in 3 minutes at 45° C.: 11.16 g.(0.041 M.) 7-ACA, 1.7 g. NaHSO₃ and 12.08 g. (0.0492 M.)1-t-butoxycarbonylaminoethyl-5-mercaptotetrazole. The nitrogen was thenshut off and the mixture was heated to 56° C., titrated to pH 6.6 withNaHCO₃ (about 8 g.), kept at 56° C. for 4 hours (pH 6.5-6.9), cooled to5° C., titrated to pH 5.0 and filtered to collect 10.09 g. (54% yield)of7-amino-3-(1-[2'-t-butoxycarbonylaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid containing about 10% of 7-ACA by NMR.

B.7-(α-Methoxyiminofuryl-2-acetamido)-3-(1-[2'-t-butoxycarbonylaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicAcid ##STR30##

Sodium α-methoxyiminofurylacetate (1.26 g.; 6.6 mM.) was suspended in 50ml. of benzene and treated with 8 drops of dry DMF. A total of 0.62 ml.(1.1 eq.) of oxalyl chloride was added and the solution was stirred for40 minutes at 20° C.

The benzene was removed at 35° C. (15 mm.) and the acid chloride (thegummy residue) was dissolved in 20 ml. acetone (NaCl insoluble). Asolution of 2.4 g. (5.25 mM).7-amino-3-(1-[2'-t-butoxycarbonylaminoethyl]-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid and 1.6 g. NaHCO₃ (pH 7.0) and 5 ml. of acetone in 40 ml. of waterwas cooled to 3° C. The acetone solution of the above acid chloride wasadded and the reaction was stirred for 40 minutes at 20° C. (pH 7.0).

The solution was titrated to pH 1.8 with hydrochloric acid, extractedthree times with 120 ml. ethyl acetate, backwashed with 100 ml. waterand azeotroped to dryness at 30° C. (15 mm.). The residue was trituratedwith 100 ml. ether, collected by filtration and dried under vacuum togive 2.38 g. (74% yield) of7-(α-methoxyiminofuryl-2-acetamido)-3-(1-[2'-t-butoxycarboxyaminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

C.7-(α-Methoxyiminofurylacetamido)-3-(1-[2'-aminoethyl]-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicAcid ##STR31##

A solution of 2.64 g. (4.34 mM.) of7-(α-methoxyiminofuryl-2-acetamido)-3-(1-[2'-t-butoxycarboxyaminoethyl]-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid in 66 ml. 97% HCOOH (40 mg./ml.) was stirred for 2 hours at 20° C.The excess HCOOH was then evaported at 30° C. (15 mm.). The residue wasazeotroped to dryness three times with 20 ml. ethyl acetate. The residuewas triturated with ether, collected by filtration and washed with etherto give 2.24 g. of7-(α-methoxyiminofurylacetamido)-3-(1-[2'-aminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (contaminated with ether and ethyl acetate and formic acid).

Analysis calculated for C₁₈ H₂₀ N₈ O₆ S₂.H₂ O[0.5 M. HCOOH; 0.5 M. ethylacetate; 0.3 M. ether]: KF(H₂ O), 2.92; C, 42.32; H, 4.90; N, 18.19; S,10.41. Found: KF(H₂ O), 1.67; C, 43.20; H, 4.38; N, 17.90; S, 10.64.

EXAMPLE 3

Substitution of an equimolar weight ofsodium-2-ethoxyimino-2-(fur-2-yl)acetate for thesodium-2-methoxyiminofuryl acetate used in the procedure of Example 1produces7-(2-ethoxyimino-2-furylacetamido)-3-(1-[2'-aminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 4

Substitution of an equimolar weight ofsodium-2-n-propoxyimino-2-(fur-2-yl)acetate for thesodium-2-methoxyiminofuryl acetate used in the procedure of Example 1produces7-(2-n-propoxyimino-2-furylacetamido)-3-(1-[2'-aminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 5

Substitution of an equimolar weight ofsodium-2-n-butoxyimino-2-(fur-2-yl)acetate for thesodium-2-methoxyiminofurylacetate used in the procedure of Example 1produces7-(2-n-butoxyimino-2-furylacetamido)-3-(1-[2'-aminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 6

The products of Examples 1-5 are prepared as syn isomers essentiallyfree of the corresponding anti isomers by the use in the procedures ofthose examples of purified syn isomers of the appropriate2-alkoxyimino-2-(fur-2-yl)-acetic acid. Conversion of part of the synisomer to anti isomer during preparation of the acid chloride from theacid is substantially avoided by minimizing its exposure to hydrogenchloride, e.g. by first converting the acid to its anhydrous sodium saltand by treating that salt with oxalyl chloride under anhydrousconditions in the presence of a hydrogen ion acceptor such asdimethylformamide.

Such syn isomers are also named as (2Z)-2-alkoxyimino-2-(fur-2-yl)aceticacids.

EXAMPLE 7

An injectable pharmaceutical composition is formed by adding sterilewater or sterile saline solution (2 ml.) to 100-500 mgm. of7-[(2Z)-2-methoxyimino-(fur-2-yl)acetamido]-3-(1-[2'-aminoethyl]tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt or formate or hydrochloride salt.

Pharmaceutical compositions of the sodium, potassium, formate andhydrochloride salts of the other compounds of the present invention,preferably in the form of the pure syn isomer, are formulated in asimilar manner.

When the compounds are first prepared in the form of the free acid theyare converted to the desired, highly water soluble potassium salt bytreatment with potassium 2-ethylhexanoate in an anhydrous solvent suchas n-butanol.

It is occasionally advantageous to have mixed with said solidcephalosporin as a stabilizing and/or solubilizing agent a sterile,anhydrous solid such as sodium carbonate, potassium carbonate or lithiumcarbonate (e.g. in about 5 or 6 percent by weight of the weight of thecephalosporin) or such as L-lysine, arginine or histidine (e.g. in about20-50% by weight of the weight of the cephalosporin) or such as asodium, potassium or calcium salt or levulinic acid, citric acid,ascorbic acid, tartaric acid or pyruvic acid (e.g. in about 25-200% byweight of the weight of the cephalosporin) or such as sodiumbicarbonate, ammonium carbamate, alkali metal or ammonium phosphates orN-methylglucamine (per U.K. No. 1,380,741).

There is also provided by the present invention a compound having theformula ##STR32## wherein R⁷ is alkyl containing 1-4 carbon atoms and Mis ##STR33## n is 0 to 4; R is hydrogen, alkyl having 1 to 8 carbonatoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, C₁ -C₄ phenalkyl,pyridyl, thienyl, or pyrrolyl;

R¹ is hydrogen, methyl or ethyl; R² and R³ are each hydrogen, alkylhaving 1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R⁴ and R⁵ areeach hydrogen or alkyl of 1 to 4 carbon atoms; R⁶ is alkyl having 1 to 4carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl,thiadiazolyl, amino or C₁ -C₄ alkylamino; X is NH or oxygen; and eachphenyl group is unsubstituted or substituted with one or twosubstituents selected from the group consisting of alkyl having 1 to 6carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy, amino, NHR¹,N(R¹)₂, nitro, fluoro, chloro, bromo or carboxy, or a nontoxic,pharmaceutically acceptable salt thereof, said compound being at least75% by weight in the form of its syn isomer and preferably in the formof its syn isomer essentially free of the corresponding anti isomer.

There is also provided by the present invention a compound having theformula ##STR34## wherein R¹ is alkyl containing 1-4 carbon atoms and R³is selected from the group consisting of ##STR35## wherein R⁵ is ahydrogen atom, a methyl or an ethyl group; X² is --O--, --NH--; R⁶ is abasic group such as alkyl or aralkyl substituted with substituted orunsubstituted NH₂, such as alkyl-NHCH₃, aralkyl-NHCH₃, ##STR36## R⁷ isan alkyl group such as a methyl, ethyl, propyl, isopropyl, butyl,isobutyl, pentyl or 2-ethylhexyl group; a cycloalkyl group such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; an arylgroup such as phenyl or naphthyl; an aralkyl group such as benzyl ornaphthylmethyl; a heterocyclic group and wherein the alkyl, cycloalkyl,aryl, aralkyl and heterocyclic groups may be substituted with one ormore groups selected from the class consisting of amino groups,substituted amino groups such as methylamino, diethylamino or acetamidogroups, the halogen groups such as fluorine, chlorine or bromine, nitrogroups, alkoxy group such as methoxy, ethoxy, propyloxy, isopropyloxy,butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable saltthereof, said compound being at least 75% by weight in the form of itssyn isomer and preferably in the form of its syn isomer essentially freeof the corresponding anti isomer.

There is also provided by the present invention a compound having theformula ##STR37## wherein R¹ is alkyl containing 1-4 carbon atoms and Mis ##STR38## wherein Y is alkyl of one to six carbon atoms, phenyl,benzyl, alkoxy of one to six carbon atoms, or benzyloxy; Z is alkyl ofone to six carbon atoms, phenylbenzyl, alkoxy of one to six carbonatoms, cyclopentyl, cyclohexyl and phenyl, or Y+Z taken together are a3-benzoxazolidine ring; or a nontoxic, pharmaceutically acceptable saltthereof, said compound being at least 75% by weight in the form of itssyn isomer and preferably in the form of its syn isomer essentially freeof the corresponding anti isomer.

Also included within the present invention are pharmaceuticalcompositions comprising a mixture of an antibacterially effective amountof a compound of the present invention and a semisynthetic penicillin oranother cephalosporin or a cephamycin or a β-lactamase inhibitor or anaminoglycoside antibiotic.

There is further provided by the present invention a pharmaceuticalcomposition comprising an antibacterially effective amount of a compoundhaving the formula ##STR39## wherein R¹ is alkyl containing 1-4 carbonatoms and R² is hydrogen, pivaloyloxymethyl, acetoxymethyl,methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl,3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic,pharmaceutically acceptable salt thereof, said compound being at least75% by weight in the form of its syn isomer and preferably in the formof its syn isomer essentially free of the corresponding anti isomer, anda pharmaceutically acceptable carrier therefor.

There is further provided by the present invention a pharmaceuticalcomposition comprising an antibacterially effective amount of the synisomer of a compound having the formula ##STR40## or a nontoxic,pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier therefor.

There is further provided by the present invention a method of treatingbacterial infections comprising administering by injection to aninfected warm-blooded animal, including man, an effective but nontoxicdose of 250-1000 mgm. of a compound having the formula ##STR41## whereinR¹ is alkyl containing 1-4 carbon atoms and R² is hydrogen,pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl,p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl or anontoxic, pharmaceutically acceptable salt thereof, said compound beingat least 75% by weight in the form of its syn isomer and preferably inthe form of its syn isomer essentially free of the corresponding antiisomer.

There is further provided by the present invention a method of treatingbacterial infections comprising administering by injection to aninfected warm-blooded animal, including man, an effective but nontoxicdose of 250-1000 mgm. of the syn isomer of a compound having the formula##STR42## or a nontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a method for combattingHaemophilus infections which comprises administering to a warm-bloodedmammal infected with an Haemophilus infection an amount effective fortreating said Haemophilus infection of a composition comprising acompound having the formula ##STR43## wherein R¹ is alkyl containing 1-4carbon atoms and R² is hydrogen, pivaloyloxymethyl, acetoxymethyl,methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl,3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic,pharmaceutically acceptable salt thereof, said compound being at least75% by weight in the form of its syn isomer and preferably in the formof its syn isomer essentially free of the corresponding anti isomer, anda pharmaceutically acceptable carrier therefor.

There is also provided by the present invention a method for combattingNeisseria infections which comprises administering to a warm-bloodedmammal infected with a Neisseria infection an amount effective fortreating said Neisseria infection of a composition comprising a compoundhaving the formula ##STR44## wherein R¹ is alkyl containing 1-4 carbonatoms and R² is hydrogen, pivaloyloxymethyl, acetoxymethyl,methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl,3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic,pharmaceutically acceptable salt thereof, said compound being at least75% by weight in the form of its syn isomer and preferably in the formof its syn isomer essentially free of the corresponding anti isomer, anda pharmaceutically acceptable carrier therefor.

Biological testing was performed according to the procedures describedby F. Leitner et al., Antimicrobial Agents and Chemotherapy 10(3),426-435 (September, 1976).

                                      TABLE 1                                     __________________________________________________________________________    Antibiotic Activity in Nutrient Broth                                                          MIC (mcg./ml.)                                                                Compound of  Compound of                                     Organism         Example 1B                                                                          Cefuroxime                                                                           Example 1B                                                                           Cefuroxime                               __________________________________________________________________________    S. pneumoniae*                                                                            (10.sup.-3)**                                                                      0.008 0.004  0.004  0.004                                    Str. pyogenes*                                                                            (10.sup.-3)**                                                                      0.008 0.004  0.004  0.004                                    S. aureus Smith                                                                           (10.sup.-4)                                                                        0.5   0.13   0.5    0.5                                      S. aureus + 50% serum                                                                     (10.sup.-4)                                                                        2     2      1      2                                        S. aureus BX1633                                                                          (10.sup.-3)                                                                        0.5   0.5                                                    S. aureus BX1633                                                                          (10.sup.-2)                                                                        1     0.5    0.5    2                                        S. aureus Meth-Res                                                                        (10.sup.-3)                                                                        2     2      >125   >125                                     Str. faecalis                                                                             (10.sup.-4)       63     >125                                     Sal. enteritidis                                                                          (10.sup.-4)                                                                        ≦0.25                                                                        0.5                                                    E. coli Juhl                                                                              (10.sup.-4)                                                                        8     4      2      8                                        E. coli     (10.sup.-4)                                                                        8     4                                                      E. coli     (10.sup.-4)       2      8                                        K. pneumoniae                                                                             (10.sup.-4)                                                                        1     2                                                      K. pneumoniae                                                                             (10.sup.- 4)                                                                       2     2      16     32                                       K. pneumoniae                                                                             (10.sup.-4)       32     125                                      Pr. mirabilis                                                                             (10.sup.-4)                                                                        0.5   0.5    0.5    0.5                                      Pr. morganii                                                                              (10.sup.-4)                                                                        2     32     1      63                                       Pr. vulgaris                                                                              (10.sup.-4)       0.5    1                                        Pr. rettgeri                                                                              (10.sup.-4)       1      16                                       Ser. marcescens                                                                           (10.sup.-4)                                                                        8     >125   8      >125                                     Ent. cloacae                                                                              (10.sup.-4)                                                                        32    >125   16     >125                                     Ent. cloacae                                                                              (10.sup.-4)                                                                        0.5   1                                                      Ent. cloacae                                                                              (10.sup.-4)                                                                        8     >125   8      >125                                     Ps. aeruginosa                                                                            (10.sup.-4)                                                                        125   >125   63     500                                      Ps. aeruginosa carb.                                                                      (10.sup.-4)       250    >1000                                    Ps. aeruginosa                                                                            (10.sup.-4)       125    500                                      Ps. aeruginosa                                                                            (10.sup.-4)       125    500                                      Ps. aeruginosa                                                                            (10.sup.-4)       63     250                                      Ps. aeruginosa                                                                            (10.sup.-4)       125    1000                                     __________________________________________________________________________     *45% AAB + 5% serum + 50% broth listed above.                                 **Dilution of overnight broth culture.                                         Cefuroxime is sodium                                                         6R,7R3-carbzmoyloxymethyl-7-(2Z)-2-methoxyimino(fur-2-yl)acetamidoceph-3-    m-4-carboxylate.                                                          

                  TABLE 2                                                         ______________________________________                                         Secondary in vitro Screening Results                                         MIC (mcg./ml)                                                                                 Compound of                                                   Organism        Example 1B   Cefuroxime                                       ______________________________________                                        Enterobacter cloacae                                                                          63           >125                                             Enterobacter aerogenes                                                                        4            4                                                Enterobacter cloacae                                                                          4            8                                                Enterobacter species                                                                          2            2                                                Escherichia coli                                                                              8            >125                                             Escherichia coli                                                                              2            4                                                Escherichia coli                                                                              2            2                                                Escherichia coli                                                                              8            32                                               Escherichia coli                                                                              4            16                                               Escherichia coli                                                                              2            4                                                Escherichia coli                                                                              2            4                                                Escherichia coli                                                                              0.5          125                                              Klebsiella species                                                                            4            8                                                Klebsiella species                                                                            2            2                                                Klebsiella species                                                                            4            4                                                Providencia stuarti                                                                           4            2                                                Proteus mirabilis                                                                             8            4                                                Proteus mirabilis                                                                             4            8                                                Proteus vulgaris                                                                              8            >125                                             Proteus vulgaris                                                                              8            >125                                             Proteus vulgaris                                                                              2            2                                                Proteus vulgaris                                                                              0.25         0.13                                             Proteus morganii                                                                              63           >125                                             Proteus morganii                                                                              4            32                                               Proteus morganii                                                                              4            16                                               Proteus morganii                                                                              4            16                                               Proteus rettgeri                                                                              0.004        0.004                                            Serratia marcescens                                                                           >63          >125                                             Serratia marcescens                                                                           4            16                                               Staphylococcus aureus                                                                         0.25         0.5                                              Staphylococcus aureus                                                                         0.5          0.5                                              Staphylococcus aureus                                                                         0.5          0.5                                              Staphylococcus aureus                                                                         0.5          0.5                                               Staphylococcus aureus                                                                        1            1                                                Staphylococcus aureus                                                                         0.5          0.5                                              Staphylococcus aureus                                                                         1            1                                                Staphylococcus aureus                                                                         1            0.5                                              Staphylococcus aureus                                                                         0.5          0.5                                              Staphylococcus aureus                                                                         0.5          0.5                                              Escherichia coli                                                                              4            4                                                Escherichia coli                                                                              2            4                                                Proteus mirabilis                                                                             2            1                                                Proteus mirabilis                                                                             4            1                                                Proteus mirabilis                                                                             4            1                                                Proteus morganii                                                                              2            2                                                Proteus mirabilis                                                                             2            1                                                Proteus morganii                                                                              .4           63                                               Proteus vulgaris                                                                              0.25         0.25                                             Proteus vulgaris                                                                              2            1                                                Proteus vulgaris                                                                              2            1                                                Proteus vulgaris                                                                              2            2                                                Proteus mirabilis                                                                             2            1                                                Proteus mirabilis                                                                             2            2                                                Enterobacter aerogenes                                                                        16           125                                              Proteus morganii                                                                              4            8                                                Proteus morganii                                                                              4            8                                                Proteus morganii                                                                              4            32                                               Proteus mirabilis                                                                             2            0.5                                              Proteus mirabilis                                                                             2            1                                                Proteus rettgeri                                                                              0.25         0.032                                            Proteus vulgaris                                                                              2            1                                                Providencia stuartii                                                                          4            2                                                Providencia stuartii                                                                          2            2                                                Providencia stuartii                                                                          1            0.5                                              Pseudomonas aeruginosa                                                                        >63          125                                              Serratia marcescens                                                                           >63          >125                                             Serratia marcescens                                                                           16           125                                              Serratia marcescens                                                                           2            16                                               Serratia marcescens                                                                           4            32                                               Serratia marcescens                                                                           16           63                                               Serratia marcescens                                                                           4            32                                               Serratia marcescens                                                                           >63          >125                                              Serratia marcescens                                                                          >63          >125                                             Klebsiella pneumoniae                                                                         2            2                                                Klebsiella pneumoniae                                                                         2            2                                                Klebsiella species                                                                            2            2                                                Klebsiella species                                                                            1            1                                                Klebsiella species                                                                            16           16                                               Klebsiella species                                                                            4            4                                                Klebsiella pneumoniae                                                                         4            4                                                Enterobacter cloacae                                                                          4            32                                               Enterobacter cloacae                                                                          32           32                                               Enterobacter aerogenes                                                                        8            125                                              Proteus rettgeri                                                                              4            8                                                Enterobacter aerogenes                                                                        4            4                                                Enterobacter aerogenes                                                                        4            8                                                Pseudomonas aeruginosa                                                                        >63          125                                              Proteus rettgeri                                                                              8            63                                               Proteus rettgeri                                                                              1            2                                                Proteus rettgeri                                                                              16           63                                               Proteus rettgeri                                                                              16           125                                              Proteus rettgeri                                                                              4            8                                                Proteus rettgeri                                                                              2            1                                                Proteus rettgeri                                                                              2            0.5                                              Proteus vulgaris                                                                              32           125                                              Proteus morganii                                                                              2            16                                               ______________________________________                                         (Cefuroxime is sodium                                                         6R,7R3-carbamoyloxymethyl-7-(2Z)-2-methoxyimino(fur-2-yl)acetamidoceph-3-    m-4-carboxylate.                                                          

                  TABLE 3                                                         ______________________________________                                         Secondary in vitro Screening Results                                         MIC (mcg./ml.)                                                                              Compound of                                                     Organism      Example 1B Ampicillin                                                                              Cefuroxime                                 ______________________________________                                        Hemophilus influenzae                                                                       0.5        0.5       1                                          Hemophilus influenzae                                                                       0.5        0.5       1                                          Hemophilus influenzae                                                                       0.5        0.25      0.5                                        Hemophilus influenzae                                                                       0.25       0.25      1                                          Hemophilus influenzae                                                                       0.25       0.13      0.5                                        Hemophilus influenzae                                                                       0.5        0.25      0.5                                        Hemophilus influenzae                                                                       0.5        0.25      0.5                                        Hemophilus influenzae                                                                       0.5        0.25      0.5                                        Neisseria gonorrhoeae                                                                       1          0.25      1                                          Neisseria gonorrhoeae                                                                       0.063      0.13      0.016                                      Neisseria gonorrhoeae                                                                       0.032      0.13      0.32                                       Neisseria gonorrhoeae                                                                       0.032      0.063     0.032                                      Neisseria gonorrhoeae                                                                       0.13       0.13      0.063                                      Neisseria gonorrhoeae                                                                       0.13       0.13      0.13                                       Neisseria gonorrhoeae                                                                       0.25       0.13      0.25                                       Neisseria gonorrhoeae                                                                       1          0.25      0.25                                       Neisseria gonorrhoeae                                                                       1          0.5       1                                          Neisseria gonorrhoeae                                                                       1          0.25      0.5                                        Neisseria gonorrhoeae                                                                       1          0.5       2                                          Neisseria gonorrhoeae                                                                       0.13       0.25      0.13                                       Neisseria gonorrhoeae                                                                       0.13       0.13      0.13                                       Neisseria gonorrhoeae                                                                       0.13       0.25      0.25                                       Neisseria gonorrhoeae                                                                       0.25       0.063     0.13                                       Haemophilus influenzae                                                                      0.5        63        0.5                                        Haemophilus influenzae                                                                      0.25       8         0.25                                       Haemophilus influenzae                                                                      0.5        63        0.5                                        Haemophilus influenzae                                                                      0.25       63        0.5                                        Haemophilus influenzae                                                                      0.5        63        1                                          Haemophilus influenzae                                                                      0.25       8         1                                          Haemophilus influenzae                                                                      0.25       0.13      0.25                                       ______________________________________                                         Cefuroxime is sodium                                                          6R,7R3-carbamoyloxymethyl-7-(2Z)-2-methoxyimino(fur-2-yl)acetamidoceph-3-    m-4-carboxylate.                                                          

                                      TABLE 4                                     __________________________________________________________________________    Mouse Blood Levels*                                                                  Blood Levels (mcg./ml.)                                                                             Sensitivity                                             Hours After (Administration                                                                         Limit                                            Compound                                                                             0.25  0.5   1    1.5  (mcg/ml.)                                                                           Solubility                                 __________________________________________________________________________    Compound of                                                                          35    17.9  5.4  <2.5 2.5   susp.                                      Example 1B                                                                           (26.6-46)                                                                           (13.4-23.9)                                                                         (4.4-6.7)                                                  Cefuroxime                                                                           34.5  26.3  7.6  3.1                                                          (28.2-42.3)                                                                         (21.4-32.4)                                                                         (6.2-9.3)                                                                          (2.2-4.6)                                                                          1.4   sol.                                       Compound of                                                                          33.6  17.4  3.9  <2.6                                                  Example 1B                                                                           (30-37.6)                                                                           (14.8-20.4)                                                                         (3.1-5.1) 2.6   sol.                                       Cefuroxime                                                                           25.6  20.6  6    2.1                                                          (19.5-35.5)                                                                         (14.6-28.9)                                                                         (4.3-8.4)                                                                          (1.5-2.8)                                                                          0.9   sol.                                       __________________________________________________________________________     *Each group consisted of 6 animals and one blood sample was taken from        each animal.                                                                  Compounds prepared in phosphate buffer and dosed IM at 40 mgm./kg.            Assay Organism: B. subtilis at pH 6.0                                         Cefuroxime is sodium                                                          6R,7R3-carbamoyloxymethyl-7-(2Z)-2-methoxyimino(fur-2-yl)acetamidoceph-3-    m-4-carboxylate.                                                          

We claim:
 1. A compound having the formula ##STR45## wherein R¹ is alkylof 1-4 carbon atoms and R² is hydrogen or a conventional,pharmaceutically acceptable, easily hydrolyzed ester forming group; or anontoxic, pharmaceutically acceptable salt thereof, said compound beingat least 75% by weight in the form of its syn isomer.
 2. The syn isomerof a compound having the formula ##STR46## wherein R¹ is alkyl of 1 to 4carbon atoms or a nontoxic, pharmaceutically acceptable salt thereof. 3.The syn isomer of the compound having the formula ##STR47## or anontoxic, pharmaceutically acceptable salt thereof.
 4. The syn isomer ofthe compound having the formula ##STR48## or a nontoxic,pharmaceutically acceptable salt thereof.
 5. The syn isomer of acompound having the formula ##STR49## wherein R¹ is alkyl of 1-4 carbonatoms and R² is hydrogen, pivaloyloxymethyl, acetoxymethyl,methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl,3-phthalidyl or 5-indanyl or a nontoxic, pharmaceutically acceptablesalt thereof.
 6. The syn isomer of a compound having the formula##STR50## wherein R¹ is alkyl of 1-4 carbon atoms.
 7. The syn isomer ofthe compound having the formula ##STR51##
 8. The syn isomer of thecompound having the formula ##STR52##